Increased accumulation of the amyloid-Beta peptide (ABeta) or specific isoforms (ABeta42) is a major pathogenic event underlying neurodegeneration in all forms of Alzheimer's disease (AD). In the first four years of this project, we have focused on the role of presenilin (PS) FAD mutations in apoptosis, and how apoptosis influences ABeta production. However, evidence has mounted to suggest that while apoptosis-induced ABeta generation may occur following acute injury to the CNS, other pathogenic mechanisms are likely involved in ABeta production owing to familial AD mutations in APP and the PS genes. Cloning of the PS genes has led to the initial characterization of the protease called gamma-secretase that cleaves APP at the C-terminal end of ABeta. Gamma-secretase is a heteromeric complex of proteins, in which only two components have been identified to date, PS and nicastrin. FAD mutations in PS1 increase the ratio of ABeta 42:ABeta40 and are likely to involve a number of as of yet unidentified proteins in the gamma-secretase/PS1. Thus, in the coming funding period, we propose to expand upon Specific Aim 3 of the original application, by exploring how the gamma-secretase complex/PS1 and FAD mutations in PS lead to alterations in the maturation and processing of APP and affect ABeta production. In our preliminary data, we show that nicastrin and thirteen unknown proteins coimmunoprecipitate with PS1 C- and N-terminal fragments from a sodium carbonate-washed lysate, thereby representing potentially novel membrane-associated components and/or substrates of the gamma-secretase/PS1 complex. We have also identified a subcellular fraction in the Golgi/endosomes harboring the complex, together with its APP C-terminal substrates. We have tentatively already identified one of the unknown protein bands in the complex. To follow up on these findings and extend our studies of the effect of FAD presenilin mutations on gamma-secretase activity, we propose to identify and characterize novel components of the gamma-secretase/PS1 complex, especially those that modulate ABeta production and the ABeta42/ABetatotal ratio. We will also test polymorphisms in genes that encode novel components of the gamma-secretase/PS1 complex, for family-based association with Alzheimer's disease. We plan to determine the subcellular localization and elucidate the physiological functions of the gamma-secretase/PS1 complex. Finally, we are performing in vitro gamma-secretase assays and reconstituting the isolated complex into unilamellar liposomes to study its activity in vitro. The overall goal of these studies is to define the pathogenetic mechanism by which more than 100 FAD mutations in PS affect ABeta generation, and to ultimately identify potential targets for reducing ABeta generation in Alzheimer's disease.